Drug-induced oxidative stress actively prevents caspase activation and hepatocyte apoptosis

Cell death is a fundamental process in health and disease. Emerging research shows the existence of numerous distinct cell death modalities with similar and intertwined signaling pathways, but resulting in different cellular outcomes, raising the need to understand the decision-making steps during cell death signaling. Paracetamol (Acetaminophen, APAP)-induced hepatocyte death includes several apoptotic processes but eventually is executed by oncotic necrosis without any caspase activation. Here, we studied this paradoxical form of cell death and revealed that APAP not only fails to activate caspases but also strongly impedes their activation upon classical apoptosis induction, thereby shifting apoptosis to necrosis. While APAP intoxication results in massive drop in mitochondrial respiration, low cellular ATP levels could be excluded as an underlying cause of missing apoptosome formation and caspase activation. In contrast, we identified oxidative stress as a key factor in APAP-induced caspase inhibition. Importantly, caspase inhibition and the associated switch from apoptotic to necrotic cell death was reversible through the administration of antioxidants. Thus, exemplified by APAP-induced cell death, our study stresses that cellular redox status is a critical component in the decision-making between apoptotic and necrotic cell death, as it directly affects caspase activity.


Fig. S2: APAP does not shift TNF-induced apoptosis to necroptosis but to necrosis.
A Western Blot of primary murine hepatocytes (PMH) treated for 2h with indicated concentrations of APAP, followed by removal of APAP (short APAP, sAPAP), and treatment with 1 ng/ml TNF plus 30 nM ActD for 12h.Numbers on the left indicate molecular weight in kDa B PMH were treated for 2h with 20 mM APAP with followed by removal of APAP and treatment with 50 µg/ml cisplatin for 16h.Results of MTT assay are shown as mean + SD of n=4.
C Schematic illustration of TNF-induced survival, apoptosis, and necroptosis via treatment with specific inhibitors such as BV6, QVD and Nec1 D Western Blot of PMH treated for 2h with 20 mM APAP with subsequent removal of APAP and treatment with 50 µg/ml cisplatin for 16h.E PMH were treated with 20 mM APAP for 2 h (sAPAP) with subsequent removal of APAP and treatment with 2 ng/ml TNF plus 5 µM BV6 for 16h, plus 30 min pre-treatment with 10 µM QVD and/or 4 µM Necrostatin-1 (Nec1).Results of MTT assay are shown as mean + SD of n=4.
Statistical significance was tested using Two-way ANOVA with Sidak's multiple comparison test.
F Representative fluorescence microscopy pictures of propidium iodide (red) and Hoechst (blue)-stained PMH treated as described in (E).Scale bar 50 µm.

Cell death
Cell death was measured by 3-(

RT-qPCR
To measure mRNA with Real-time quantitative PCR (RT-qPCR), hepatocytes or liver pieces were lysed in TRIzol TM Reagent (Invitrogen) and RNA was isolated according to manufacturer's

ATP, GSH, and ROS Assays
Cellular ATP, GSH and ROS levels were measured with the CellTiter-Glo® 2.0 Assay, GSH/GSSG-Glo TM Assay or the ROS-Glo TM H2O2 Assay according to the manufacturer's protocols (Promega).Notably, APAP-induced cell death was not yet present at the assay end points.

Sample processing for mass spectrometric (MS) analysis
To analyze post-translational modifications, isolated hepatocytes were seeded in 15 cm dishes and treated on the following day with and without 20 mM APAP for 4 h before they were lysed in CHAPS Buffer (50 mM Tris, 150 mM NaCl, 0.5% deoxycholic acid, 1% CHAPS, pH 8.0) for 30 min on ice.Lysates were cleaned by centrifugation at 14 000 xg for 15 min at 4 °C.Protein concentration of cleaned lysates were between 4-6 µg/ml.Samples were prepared in four biological replicates.Proteins were precipitated by addition of 6 volumes of ice-cold acetone and incubated for 15 min at -80 °C, followed by 90 min at -20 °C.Precipitated proteins were pelleted by centrifugation at 16 000 xg for 10 min at 4 °C, and completely dried before pellets were resuspended in 8M urea.To retain reversible cysteine modifications, reduction and alkylation of cysteine residues was omitted.Urea concentration was diluted to 4M, before a Trypsin/Lys-C mix (Promega, V5071) at an enzyme-substrate ratio of 1:100 was added.Lys-C digestion was allowed to proceed for 3 h at 37°C before samples were further diluted to 1M urea, and trypsin digestion was conducted for 18 h at 37 °C.Digestion was stopped by addition of formic acid to a final concentration of 2 % (v/v).Peptides were desalted using C18 Spin Tips (Pierce, #84850), dried by vacuum centrifugation and stored at -20 °C prior to MS analysis.

Mass spectrometric analysis -Data independent acquision
Roughly 500 ng peptides were resuspended in 5 % ACN, 0.1 % formic acid and loaded onto a QExactive HF mass spectrometer coupled with an EASY-nLC 1200 system (both Thermo Scientific) equipped with a 50 cm Acclaim PepMap C18 column (Thermo Scientific, P/N 164945).Peptides were separated across a 220 min gradient with a flow of 150 nL/min at 50 °C oven temperature.MS was operated in data independent acquision (DIA) in variable window mode using 24 windows 6 .Full mass spectra were acquired in the Orbitrap at a resolution of 120 K, an AGC target of 3e6 and 60 ms maximum injection time (max.IT) in a scan range of 300-1650 m/z.DIA scans were acquired in the Orbitrap at a resolution of 30K, an AGC target of 1e6 and max IT set to auto.Precursors were fragmented with HCD at 28 NCE.

Mass spectrometric analysis -Parallel reaction monitoring
Parallel reaction monitoring (PRM) analysis was conducted on an Orbitrap Tribrid Fusion mass spectrometer connected to an EASY-nLC1200 liquid chromatography system (both Thermo Scientific).Peptides were separated across an 80 min gradient at 300 nL/min on a 15 cm Acclaim PepMap C18 column (Thermo Scientific, P/N 164943).Full mass spectra were acquired in the Orbitrap at 120 K resolution with an AGC target of 4e5 and 60 ms max.IT in a scan range of 295-1450 m/z.Precursors were isolated in the quadrupole with an isolation width of 1.6 m/z and fragmented with HCD at 28 NCE.PRM scans were acquired in the Iontrap in rapid scan mode with a defined scan range of 150-1250 m/z, an AGC target of 1.5e4 and max.
IT set to auto 7 .Loop control was set to all.The Isolation list of all precursors of interest was prepared in Skyline and set to include all peptides of the indicated proteins (P70677, Q8C3Q9, O88879, Q91VS7, O35490, Q9D172, P62897) that contained a cysteine with the defined modifications (NAPQI, Glutathione, trioxidation, nitrosylation).To minimise cycle time, this list was split into four separate lists which were used for four consecutive measurements.

Data analysis -Data obtained in DIA mode
Data acquired by DIA was analysed in Spectronaut (v.18.6.231227.55695) in directDIA mode.
BGS Factory settings were used with minimum peptide length set to 6 amino acids and variable modifications of cysteine residues to include oxidation, dixodation, trioxidation, Glutathione, Nitrosyl and NAPQI.Spectra were searched against the Uniprot database of Mus musculus (downloaded 2023/11/17).In the quantification tab, normalization filters to keep all cysteine
DEVDase AssayTo measure Caspase 3 activity, treated cells were washed with PBS and lysed Lysis Buffer (10 mM HEPES, 42 mM KCl, 5 mM MgCl2 Hexahydrate, 0.1 mM EDTA, 0.1 mM EGTA, 0.5% CHAPS, 1 mM DTT) for 5 min on ice and for 5 min at 37 °C while shaking at 1000 rpm.Lysates were centrifuged for 10 min at 4 °C, 14000 xg, and supernatant was transferred in triplicates into black 96-well plate.Reaction Buffer (25 mM HEPES, 1 mM EDTA, 0.1% CHAPS, 3 mM DTT) including 20 µM caspase-3 substrate Ac-DEVD-AFC (Enzo Life Sciences).Fluorescence was measured as kinetic (36x10 min) at 505 nm (ex: 400 nm) with the Infinite® 200 PRO plate reader (TECAN).Activity was calculated as slope in the linear range and afterwards normalized to respective protein concentration determined by Bradford Protein Assay.Data was normalized to untreated control.Western Blot and cytosolic and mitochondrial fractionsTreated cells or liver pieces were lysed in RIPA Buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, fresh 1x cOmplete™ Protease Inhibitor Cocktail (Roche)).for10minat4°C, 1000xg.The pellet containing mitochondria was washed once and then lysed in mitochondrial lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.2% Triton-X-100, 0.3% NP-40, 0.5% deoxycholic acid, 1x cOmplete™ Protease Inhibitor Cocktail (Roche)) on ice for 10 min.Mitochondrial faction was collected byImmunoprecipitationTo study protein interaction, cells were harvested from 10 cm dishes, washed and lysed in CHAPS buffer (50 mM Tris, 150 mM NaCl, 0.5% deoxycholic acid, 1% CHAPS, 1x cOmplete™ Protease Inhibitor Cocktail (Roche), pH 8.0) on ice for 30 min.As input control, 10% of centrifuged lysate was directly boiled in 1x Laemmli buffer as described.Remaining lysate was incubated with bait or isotype antibodies for 2-3h at room temperature under constant rotation.Protein G Sepharose 4 Fast Flow beads (GE Healthcare Life Sciences) were used to immunoprecipitate bound proteins after overnight incubation at 4 °C under constant rotation.Washed beads with bound proteins were boiled in 2x Laemmli buffer at 95°C for 10 min to release bound proteins.Equal volumes were used for Western Blotting.